The
emergence of SARS-CoV-2 has resulted in over 90,000 infections and over 3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into
cells and are the main target of antibodies. We show that SARS-CoV-2 S
uses ACE2 to enter cells and that the receptor-binding domains of
SARS-CoV-2 S and SARS-CoV S bind with similar affinitiesto human ACE2,
correlating with the efficient spread of SARS-CoV-2 among humans.
We
found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site
at the boundary between the S1/S2 subunits, which
is processed during biogenesis and sets this virus apart from SARS-CoV
and SARS-related CoVs.
We determined cryo-EM structures of the
SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of
vaccines and inhibitors of viral entry.
Finally, we demonstrate that
SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S
mediated entry into cells, indicating that cross-neutralizing antibodies
targeting conserved S epitopes can be elicited upon vaccination.
Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humanssince the beginning of the 21st century: severe acute respiratory syndrome coronavirus (SARS-CoV) Drosten et al., 2003,Ksiazek et al., 2003), Middle-East respiratory syndrome coronavirus (Zaki et al., 2012) (MERS-CoV), and SARS-CoV-2 (Huang et al., 2020,Zhu et al., 2020).
SARS-CoV emerged in the Guangdong province of China in 2002 and spread
to five continents through air travel routes, infecting 8,098 people and
causing 774 deaths. In 2012, MERS-CoV emerged in the Arabian Peninsula,
where it remains a major public health concern, and was exported to 27
countries, infecting a total of ∼2,494 individuals and claiming 858
lives. A previously unknown coronavirus, named SARS-CoV-2, was
discovered in December 2019 in Wuhan, Hubei province of China and was
sequenced and isolated by January 2020 (
Zhou et al., 2020,Zhu et al., 2020).
SARS-CoV-2 is associated with an ongoing outbreak of atypical pneumonia
(Covid-2019) that has affected over 90,000 people and killed more than
3,000 of those affected in >60 countries as of March 3, 2020. On
January 30, 2020, the World Health Organization declared the SARS-CoV-2
epidemic a public health emergency of international concern.
MERS-CoV was suggested to originate from bats, but the reservoir host
fueling spillover to humans is unequivocally dromedary camelsHaagmans et al., 2014,Memish et al., 2013).
Both SARS-CoV and SARS-CoV-2 are closely related and originated in
bats, who most likely serve as reservoir host for these two viruses (Ge et al., 2013,Hu et al., 2017, Li et al., 2005b,Yang et al., 2015a, Zhou et al., 2020).
Whereas palm civets and racoon dogs have been recognized as
intermediate hosts for zoonotic transmission of SARS-CoV between bats
and humans (Guan et al., 2003,Kan et al., 2005, Wang et al., 2005),
the SARS-CoV-2 intermediate host remains unknown. The recurrent
spillovers of coronaviruses in humans along with detection of numerous
coronaviruses in bats, including many SARS-related coronaviruses
(SARSr-CoVs), suggest that future zoonotic transmission events may
continue (Anthony et al., 2017,Ge et al., 2013,Hu et al., 2017, Li et al., 2005b, Menachery et al., 2015, Menachery et al., 2016,Yang et al., 2015a, Zhou et al., 2020).
In addition to the highly pathogenic zoonotic pathogens SARS-CoV,
MERS-CoV, and SARS-CoV-2, all belonging to the β-coronavirus genus, four
low-pathogenicity coronaviruses are endemic in humans:
HCoV-OC43,
HCoV-HKU1, HCoV-NL63, and HCoV-229E.
To date, no therapeutics or
vaccines are approved against any human-infecting coronaviruses.
(SPIKE Glycoprotein S) ( Kyse on niistä viruksen pinnalla kuvissa näkyvistä "miinan tapeista", jotka ovat saaneet uusia piirteitä)
Coronavirus entry into host cells is mediated by the transmembrane (TM) spike
(S) glycoprotein that forms homotrimers protruding from the viral
surface (Tortorici and Veesler, 2019).
S comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit).
As a result, coronavirus entry into susceptible cells is a complex
process that requires the concerted action of receptor-binding and
proteolytic processing of the S protein to promote virus-cell fusion.
Different coronaviruses use distinct domains within the S1
subunit to recognize a variety of attachment and entry receptors,
depending on the viral species.
* Endemic human coronaviruses OC43 and
HKU1 attach via their S domain A (SA) to 5-N-acetyl-9-O-acetyl-sialosides found on glycoproteins and glycolipids at the host cell surface to enable entry into susceptible cells Hulswit et al., 2019, Tortorici et al., 2019,Vlasak et al., 1988).
* MERS-CoV S, however, uses domain A to recognize non-acetylated sialoside attachment receptors (Li et al., 2017,Park et al., 2019), which likely promote subsequent binding of domain B (SB) to the entry receptor, dipeptidyl-peptidase 4 (Lu et al., 2013,
As
the coronavirus S glycoprotein is surface-exposed and mediates entry
into host cells, it is the main target of neutralizing antibodies (Abs)
upon infection and the focus of therapeuticand vaccine design.
We previously characterized potent human-neutralizing Abs fromrare memory B cells of individuals infected with SARS-CoV (Traggiai et al., 2004) or MERS-CoV (Corti et al., 2015)
in complex with SARS-CoV S and MERS-CoV S to provide molecular-level
information of the mechanism of competitive inhibition of SB attachment to the host receptor (Walls et al., 2019).
The S230 anti-SARS-CoV Ab also acted by functionally mimicking receptor
attachment and promoting S fusogenic conformational rearrangementsthrough a ratcheting (rachet= pidättävä haka, säppiryörä) mechanism that elucidated the unique nature of the
coronavirus membrane fusion activation (Walls et al., 2019).
*We
report here that ACE2 could mediate SARS-CoV-2 S-mediated entry into
cells, establishing it as a functional receptor for this newly emerged
coronavirus. The SARS-CoV-2 SB engages human ACE2 (hACE2) with comparable affinity to SARS-CoV SB
from viral isolates associated with the 2002–2003 epidemic (i.e.,
binding with high affinity to hACE2). Tight binding to hACE2 could
partially explain the efficient transmission of SARS-CoV-2 in humans, as
was the case for SARS-CoV.
We identified the presence of an unexpected
furin cleavage site at the S1/S2 boundary of
SARS-CoV-2 S, which is cleaved during biosynthesis—a novel feature
setting this virus apart from SARS-CoV and SARSr-CoVs. Abrogation (kumoaminen, poisto) of
this cleavage motif moderately affected SARS-CoV-2 S-mediated entry into
VeroE6 or BHK cells ( kopeutkisoluja) but may contribute to expand the tropism of this
virus, as reported for several highly pathogenic avian influenza viruses
and pathogenic Newcastle disease virus (Klenk and Garten, 1994,Steinhauer, 1999).
We determined cryoelectron microscopy (cryo-EM) structures of the
SARS-CoV-2 S ectodomain trimer and reveal that it adopts multiple SB
conformations that are reminiscent of previous reports on both SARS-CoV
S and MERS-CoV S.
Finally, we show that SARS-CoV S mouse polyclonal
sera potently inhibited entry into target cells of SARS-CoV-2 S
pseudotyped viruses.
Collectively, these results pave the way for
designing vaccines eliciting broad protection against SARS-CoV-2,
SARS-CoV, and SARSr-CoV.
Results
ACE2 Is an Entry Receptor for SARS-CoV-2
Figure 1ACE2 Is a Functional Receptor for SARS-CoV-2 S
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